1. Field of the Invention
The present invention relates to a method for identifying a mutation or a polymorphism in a nucleic acid sequence. The present invention is particularly useful for diagnoses of genetic diseases, analyses of nucleotide polymorphisms and the like.
2. Description of Related Art
As used herein, a nucleotide polymorphism refers to a difference in a nucleotide sequence from a wild type. It is known that nucleotide polymorphisms of genes play important roles in drug metabolism as causal factors of variation among individuals in side effects or unsuccessful therapies. They are also known as causal factors of individual differences known as constitutions such as basal metabolism. In addition, nucleotide polymorphisms serve as genetic markers for a number of diseases. Therefore, it is clinically important to elucidate such mutations. Routine phenotypic classification is particularly recommended for psychiatric patients and suicidal patients in clinical studies (Gram and Brsen, European Consensus Conference on Pharmacogenetics. Commission of the European Communities, Luxembourg, pp. 87-96 (1990); Balant et al., Eur. J. Clin. Pharmacol., 36:551-554 (1989)). A method for analyzing a nucleic acid sequence is desired for detection of genotypes following identification of a responsible mutant type gene.
A nucleic acid sequence determination method (a sequencing method) exemplifies a conventional nucleic acid sequence analysis technique. A sequencing method may be used to detect and identify a nucleotide polymorphism contained in a nucleic acid sequence. However, it requires a great deal of labor and time for preparation of a template nucleic acid, a DNA polymerase reaction, polyacrylamide gel electrophoresis, analysis of a nucleic acid sequence and the like. The labor may be saved by using a recent automated sequencer, although the necessary equipment is expensive.
On the other hand, various genetic diseases are known to be caused by point mutations in genes. For some of them, the sites and types of point mutations in the genes that cause the genetic diseases are known.
A method for detecting a point mutation in a gene utilizing a gene amplification method such as the Polymerase Chain Reaction (PCR) method (JP-B 4-67960; JP-B 4-67957) is conventionally known as a method for detecting such an expected point mutation. An oligonucleotide for a wild type which is completely complementary to a terminal portion of a region to be amplified in a wild type gene, and an oligonucleotide for a mutant type which is completely complementary to a terminal portion of a region to be amplified in a mutant type gene are used as one of paired oligonucleotides for gene amplification in this method. The oligonucleotide for a mutant type has a nucleotide complementary to the nucleotide responsible for the expected point mutation at its 3′ end. A gene in a sample is subjected to a gene amplification method using such oligonucleotides for a wild type and a mutant type independently.
If a gene in a sample is a wild type, nucleic acid amplification occurs using an oligonucleotide for a wild type. However, an extension reaction does not occur and, therefore, the nucleic acid amplification does not occur using an oligonucleotide for a mutant type because the 3′ end of the oligonucleotide is not complementary to (is mismatched with) the corresponding nucleotide in the gene in the sample. On the other hand, if a gene in a sample is a mutant type, amplification does not occur using an oligonucleotide for a wild type, whereas the amplification occurs using an oligonucleotide for a mutant type. Thus, it is possible to determine whether a gene in a sample is a wild type or a mutant type by examining whether or not amplification occurs using one of the oligonucleotides, thereby identifying a point mutation in the gene in the sample. In this case, the presence of an amplified nucleic acid can be detected to examine the occurrence of amplification by electrophoresis of an amplification product on agarose gel, staining the agarose gel with a fluorescent reagent that specifically binds to nucleic acids (e.g., ethidium bromide) and UV irradiation. Alternatively, a Southern blotting method in which an amplified nucleic acid is immobilized on a nylon membrane and detection is carried out using a labeled probe; a sandwich hybridization method in which an amplified nucleic acid is captured by a capture probe immobilized on a solid substrate and a detection probe is then allowed to act thereon for detection; and the like have been developed.
Instead of direct detection of a fluorescence intensity, the Fluorescence Resonance Energy Transfer (FRET) method is utilized for detection of hybridization with a probe according to a recently developed detection method. Fluorescence resonance energy transfer is generated between a donor fluorescent substance and a quencher dye (which may be or may not be a fluorescent substance). It is generated if the absorption spectrum for one (a quencher) overlaps with the emission spectrum for another (a donor), and the two dyes are located in close proximity. A pair of dyes having such properties are called a donor/quencher dye pair or an energy transfer dye pair.
Excitation energy of a donor fluorescent substance is transferred to an adjacent quencher due to a dipolar interaction caused by a resonant dipole, resulting in quenching of the donor fluorescent substance. In some cases, if the quencher is also a fluorescent substance, the fluorescence intensity may be enhanced. An energy transfer efficiency highly depends on the distance between the donor and the quencher. An equation for estimating the relationship was developed by Forster (Forster, T., Ann. Phys., 2:55-75 (1948)). A distance between a donor and a quencher dye that results in an energy transfer efficiency of 50% is called a Forster distance (Ro). For example, other modes of fluorescence quenching include charge transfer quenching and collisional quenching.
Energy transfer and other modes based on quenching caused by an interaction between two adjacent dyes can be conducted in a homogeneous manner. Therefore, it is an attractive means of detecting or identifying a nucleotide sequence. A homogeneous analytical system is simpler than a conventional method of analyzing hybridization with a probe which is based on detection of fluorescence from a single fluorescent label. This is because a heterogeneous analysis generally requires a further step of separating hybridized labels from free labels which are not hybridized. Typically, the FRET and other related methods are based on monitoring of a change in a fluorescence profile of one (or both) of dye labels upon binding by hybridization of two complementary oligonucleotides. The change in a fluorescence profile is measured as a change in an energy transfer level or a change in a fluorescence quenching level in this system. Typically, it is expressed as an increase in a fluorescence intensity of one of the dyes.
A nucleotide sequence of interest can be detected without separating hybridized oligonucleotides from unhybridized oligonucleotides according to this method. Hybridization can be carried out using two separate complementary oligonucleotides, one labeled with a donor fluorescent substance and another with a quencher.
A nucleotide polymorphism can be identified by using an oligonucleotide having a nucleotide polymorphism-specific sequence as one of the oligonucleotides to provide a polymorphism-specific FRET signal. Several systems for methods of analyzing FRET hybridization are reviewed by Kricka, L. J. (ed.), “Nonisotopic DNA Probe Techniques”, Academic Press Inc., pp. 311-352 (1992). Alternatively, one can attach a donor and a quencher to a single oligonucleotide such that the presence and absence of hybridization of the oligonucleotide to a complementary sequence result in a detectable difference in one (or both) of fluorescence profiles. Typically, if an oligonucleotide is hybridized, donor fluorescence is increased and energy transfer/quenching is decreased in this system. For example, if a self-complementary oligonucleotide having labels at both ends forms a hairpin structure, the two fluorescent substances at the 5′ end and the 3′ end are located in close proximity, resulting in energy transfer and quenching. If the self-complementary oligonucleotide is hybridized to a complementary sequence in a second oligonucleotide, the hairpin structure is destroyed and the distance between the two dyes is increased. Then, the quenching is decreased.
The drawbacks associated with the hairpin structure are as follows. Since the stability is high, conversion to a non-quenching hybridization form is often slow. The performance is generally low with only little inclination towards the non-quenching hybridization form. A hairpin structure labeled as described above that contains a sequence for detection in a loop between self-complementary arms forming a stem is described in Tyagi, S. and Kramer, F. R., Nature Biotechnol., 14:303-308 (1996). The base-paired stem must be melted for hybridization between the sequence for detection and a target which causes decrease in quenching. A “double hairpin” probe and a method of utilizing the same are described in B. Bagwell et al. (Nucleic Acids Res., 22:2424-2425 (1994); U.S. Pat. No. 5,607,834). A target-binding sequence is contained within the hairpin structure. Competitive hybridization participates between the target and a self-complementary sequence in the hairpin structure. Bagwell et al. solve the problem of disadvantageous hybridization kinetics by destabilizing the hairpin structure using a mismatch.
It may be possible to identify a nucleotide polymorphism using this method by selecting a nucleotide polymorphism-specific sequence as a target-binding sequence. It is generally necessary to select stringent hybridization conditions in order to distinguish a difference in a single nucleotide using an oligonucleotide hybridization method.
A homogeneous method in which energy transfer or another fluorescence quenching mode is utilized for detection of nucleic acid amplification has been reported. A real-time detection method in which a doubly labeled detection probe is cleaved in a target amplification-specific manner during a PCR is disclosed in L. G. Lee et al. (Nucleic Acids Res., 21:3761-3766 (1993)). If a detection probe is hybridized to a portion downstream from an amplification primer, the detection probe is digested by a 5′-3′ exonuclease activity of Taq polymerase. Then, the two fluorescent dyes forming an energy transfer pair are separated from each other.
It may be also possible to identify a nucleotide polymorphism by using a nucleotide polymorphism-specific probe as a doubly labeled detection probe according to this method.
A method in which detection is carried out using two probes, i.e., a labeled polymorphic site-specific probe and a probe having a donor label which is adjacent to the labeled polymorphic site-specific probe is also known (Reischl, U. et al. (eds.), “Rapid Cycle Real-Time PCR—Methods and Applications, Springer Verlag, pp. 91-96 (2001)). In this case, it is possible to identify a polymorphism by detecting two kinds of FRET signals using the label of the polymorphic site-specific probe and the donor label in combination. According to this method, it is also necessary to select stringent hybridization conditions in order to distinguish a difference in a single nucleotide using an oligonucleotide hybridization method as described above. In addition, four labeled probes are required for the method. An alternative method in which a single labeled polymorphic site-specific probe is hybridized with a donor labeled probe, the temperature is then elevated, and a polymorphism is identified based on the temperature at which the polymorphic site-specific probe is separated into a single strand is known (Bernard, P. S., et al., Anal. Biochem., 255:101-107 (1998)). However, this method requires use of two probes, i.e., a labeled polymorphic site-specific probe and a donor labeled probe, as well as control of dissociation of the donor probe into a single strand.
A method for detecting a target nucleic acid based on FRET between an intercalator as a nucleic acid-specific label and a fluorescent label of a probe hybridized with the target nucleic acid has been disclosed (Heller, “Rapid Detection and Identification of Infectious Agents”, Academic Press Inc., pp. 245-256 (1985); Cardullo, R. A. et al., Proc. Natl. Acad. Sci. USA, 85:8790-8794 (1988)); Morrison et al., in Kricka, L. J. (ed.), “Nonisotopic DNA Probe Techniques”, Academic Press Inc., Chapter 13 (1992). However, Morrison et al. report that nonspecific binding of an intercalator to portions other than the portion of hybridization with a probe results in increased noise.
A method of detection based on FRET using an intercalator SYBR Green and a probe labeled with a fluorescent label Cy5 has been disclosed (WO 99/28500). However, it has been reported that the use of SYBR Green and Cy5-labeled probe in FRET results in high noise and, therefore, is not suitable for practical use (Cane, P. A. et al., Antimicrob. Agents Chemother., 43:1600-1608 (1999)).
One may consider that a polymorphism can be readily identified by detecting an amplified nucleic acid according to a method as described above. However, the conventional methods have problems for practical use as follows. Since the procedures of the methods are complicated, analyses by obtaining stable signals for a wild type and a polymorphism are made difficult, and a great deal of works are required in order to identify a polymorphism accurately.
For example, in case of electrophoresis, it is necessary to detect a wild type and a polymorphism separately, and it is difficult to numerically express an amount of a nucleic acid based on an electrophoretic image accurately. In case of a Southern blotting method or a sandwich hybridization method, it is necessary to strictly adjust conditions for a hybridization reaction with a probe which is required in the method. In addition, the method requires a step of removing excess probes. Thus, the operation is very complicated.
A method utilizing FRET enables a homogeneous analysis. The detection can be readily carried out because it does not require the removal of excess probes and the like. However, it has been considered doubtful if such a method can be practically used for detection of a genetic polymorphism for the following reasons. The method requires two oligonucleotide probes having different labels, or a doubly labeled probe, specific for each polymorphism. Furthermore, it has been reported that the disclosed method using SYBR Green and a CyS-labeled probe is not suitable for practical use because of the high noise as described above.
The main object of the present invention is to solve the above-mentioned problems and to provide a method that enables definite and reproducible detection of a polymorphism in a nucleic acid sequence as well as a reagent for the method.